Loken and Terstappen have previously described in U.S. Pat. No. 5,047,321, the multiparameter analysis of cellular components in a body fluid comprised of blood and bone marrow. These authors were able to discriminate among various cellular components of blood and bone marrow, count the number of cells within each component and provide a differential analysis of each of them using a combination of two nucleic acid dyes—the LDS-751 (Exciton) DNA-dye, the thiazol orange (TO, Molecular Probes, Inc) RNA-dye—, a fluorescently labeled anti-CD45 monoclonal antibody and two light scatter parameters (forward and sideward light scatter). This approach allowed the identification and differention between nucleated red cells, erythrocytes, reticulocytes, platelets, lymphocytes, monocytes, neutrophil granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells. Despite this, they could not show that with this approach they were capable of specifically differentiate between normal and neoplastic cells coexisting in the same sample or of further characterize these cell subsets.
In U.S. Pat. No. 6,287,791, Terstappen and Chen described a further refinement of the U.S. Pat. No. 5,047,321, but they did not show any better characterization of the different leukocyte populations.
In U.S. Pat. No. 0,238,009,8.0, Orfao described a procedure for the multidimensional leukocyte differential analysis of blood, bone marrow and other body fluids, which specifically allowed further identification of dendritic cells and their subsets, in addition to nucleated red cells, lymphocytes, monocytes, neutrophil granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells.
In turn, in U.S. Pat. No. 5,538,855, Orfao described a procedure that allowed a more detailed analysis of the lymphoid compartments through the simultaneous identification of up to 12 different subsets of T, B and Nk-cells in blood and bone marrow among other types of biological samples. The author used a combined staining for the CD3, CD19, CD56 (and/or CD16), CD4 and CD8 antigens in a 3-color single staining. However, by using this approach he was not able to further characterize the identified cell subsets, at the same time, not all subsets of non-lymphoid cells present in a normal peripheral blood could be specifically identified (e.g.: subpopulations of dendritic cells).
In none of the referred procedures, the methods described allowed directly for a more detailed characterization of the cells identified. In such case, the use of a greater number of stainings associated with distinguishable fluorescence emissions and of flow cytometer instruments capable of measuring a higher number of different fluorescence emissions, are required. In the past two decades, the number of different fluorescence emission that can be simultaneously measured by a flow cytometer has notably increased moving from 3 to up to 17 colors. However, this number is still limited since frequently more than 20 markers are required for a detailed characterization of the cellular components present in different normal and pathological samples. As an example, assessment of the TCRVbeta repertoire of peripheral blood TCRalfa-beta+ T-cells, typically requires staining for more than 20 different markers directed against different members of the TCRVbeta families of proteins; similarly, routine diagnostic immunophenotypic analysis of leukemic samples is based on the evaluation of the staining patterns of bone marrow and/or blood cells for up to several tenths of different markers (typically between 20 and 40 antigens). Thus, even with the ability of simultaneously measuring 17 different fluorescence emissions, the most advanced flow cytometry instruments still have limited multicolor capabilities. As mentioned above, the maximum number of simultaneously measured fluorescence emissions for an individual event or for a group of events contained in a flow cytometry data file, depends on the availability of fluorochromes with compatible, distinguishable fluorescence emissions and on the maximum number of fluorescence emissions that can be detected in the flow cytometer. Because of such limitation, staining of a sample for a number of markers higher than the number of fluorescence emissions that the available flow cytometer instrument is capable of measuring is usually done in two or more separate aliquots measured one after each other. As an example of such strategy, in U.S. Pat. No. 5,137,809, Loken and Sha described a procedure for a multiparameter analysis of cellular components in bone marrow. The authors described the use, in a first step, of a combination of monoclonal antibodies each labeled with a different fluorochrome, to stain all leukocytes and of further combinations to stain selected populations of leukocytes, in a second step. However, such approaches do not allow to automatically link and directly compare the information on the amount of light scatter and fluorescence emissions measured for individual cells contained 1) in different aliquots from the same sample, 2) in different samples derived from identical or different tissues from the same individual or from different individuals, or; 3) in different sample aliquots that have been measured under different conditions.
All procedures described above allowed the identification of a variable number of different populations of normal leukocytes present in blood, bone marrow and other samples and they only allow the identification of selected subpopulations of cells depending on the specific combination of monoclonal antibodies and nucleic acid dyes used; nevertheless, they were not able to provide an approach for the specific and reproducible identification of neoplastic cells admixtured naturally or artificially with normal cells in a sample.
In U.S. provisional patent No. 10/791.994, Orfao, Pedreira and Sobral da Costa described a procedure for the multidimensional detection of aberrant phenotypes in neoplastic cells to be used to discriminate them from normal cells, for monitoring minimal numbers of neoplastic cells in blood, bone marrow spinal fluid and lymph node samples, using flow cytometry measurements. This procedure also allows an objective comparison between flow cytometric data acquired in different measurements which corresponded to: 1) different aliquots from the same sample, 2) different aliquots from distinct samples from the same subject and 3) different aliquots from distinct samples from different individuals, even in cases where they were measured under different conditions. With this procedure, large flow cytometry data files are generated by merging data from two or more different data files containing data from one or more samples. In these data files, information about an infinite number of parameters can be stored together; however, each event in the list mode data file is only associated to data derived from the parameters actually measured in the flow cytometer for that particular event and no link can be made for individual events between the parameters that have been measured and those that have not been measured for that particular event, even if those parameters were evaluated for other events contained in the fused data file.